Compositions for Gel Production and their Uses

Abstract

The present invention provides a gel manufacturing composition that yields a gel exhibiting excellent strength. [Solution] The gel manufacturing composition according to the present disclosure comprises a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion.

Description

[Technical Field]

[0001] This invention relates to a composition for gel production and its use. [Background technology]

[0002] Injectable gels are a type of biomaterial that is attracting attention because they are administered into the body as a liquid and gel under specific conditions. Such injectable gels have a variety of applications in fields such as tissue regeneration, localized and sustained drug delivery, orthopedics, regenerative medicine, and cosmetic surgery.

[0003] Recently, polyion complexes (PICs) have become known as a technology that can be used for such injectable gels. For example, the polyion complex described in Patent Document 1, which includes a polycationic polymer and a polyanionic polymer, forms stable micelles in an aqueous medium, and an aqueous solution of such micelles gels at around body temperature. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2016 / 167333 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, the conventional techniques described above had room for further improvement in terms of the strength of the resulting gel. One aspect of the present invention aims to realize a gel manufacturing composition that can produce a gel exhibiting excellent strength. [Means for solving the problem]

[0006] To solve the above problems, a gel manufacturing composition according to one aspect of the present invention comprises a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. [Effects of the Invention]

[0007] According to one aspect of the present invention, a gel manufacturing composition can be provided that can produce a gel exhibiting excellent strength. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows the GPC curves for P1-P4 (solid lines) and the GPC curve for PEG macroinitiators (dashed lines). [Figure 2] This figure shows the 1H NMR spectra of P4 before and after deprotection. [Figure 3] This figure shows the DLS measurement results (P1-P4) for self-assembled nanoparticles (NanoLys / PSS). [Figure 4] This figure shows the DLS measurement results, transmission electron microscope (TEM) images, and 1H NMR spectra (including the 1H NMR spectrum of NaPSS) of self-assembled nanoparticles (NanoLys / PSS(P2)). [Figure 5] This figure shows the effect of NaCl concentration on the temperature dependence of the elastic modulus and viscosity measurements of self-assembled nanoparticles (NanoLys / PSS(P4)). [Figure 6] It is a diagram showing the influence of the polymerization degree of lysine on the temperature dependence measurement results of the elastic modulus of self-assembled nanoparticles (NanoLys / PSS). [Figure 7] It is a diagram showing the DLS measurement results of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)). [Figure 8] It is a diagram showing the DLS measurement results of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-30)). [Figure 9] It is a diagram showing the temperature dependence measurement results of the elastic modulus and viscosity of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)) (influence of NaCl concentration). [Figure 10] It is a diagram showing the temperature dependence measurement results of the elastic modulus and viscosity of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4)) (influence of silica nanoparticle size). [Figure 11] It is a diagram showing the DLS measurement results of RIG and RIG / ST-XS (influence of adding silica nanoparticles). [Figure 12] It is a diagram showing the temperature dependence measurement results of the elastic modulus and viscosity of RIG and RIG / ST-XS (influence of adding silica nanoparticles).

Mode for Carrying Out the Invention

[0009] An embodiment of the present invention will be described below, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A~B" representing a numerical range means "A or more and B or less".

[0010] 〔1. Composition for Gel Production〕 A gel-making composition according to one embodiment of the present invention comprises a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. Hereinafter, polyethylene glycol will be abbreviated as PEG and polyion complex as PIC. The gel-making composition may be a sol containing PIC and inorganic nanoparticles.

[0011] The aforementioned PIC forms micelles by self-assembly in an aqueous medium. Such micelles gel in response to temperature. The inventors have found that when inorganic nanoparticles are compounded into these micelles, gelation occurs in response to temperature and ionic strength, and the strength of the resulting gel is superior to that of gels without inorganic nanoparticles. While gels without inorganic nanoparticles often lack sufficient mechanical strength and become fluid after administration into the body, making them impractical, the gel obtained using the gel-making composition according to one embodiment of the present invention has sufficient strength to remain in the body. Furthermore, it has been confirmed that the gel obtained by compounding inorganic nanoparticles in this way becomes an irreversible gel that does not return to its original liquid state after gelation. It is also expected that the drug-carrying capacity will be improved by the inorganic nanoparticles.

[0012] In other words, the gel manufacturing composition and the gel can be used as an injectable gel that is administered into the body as a liquid and gels in response to body temperature and ionic strength within the body. Such an injectable gel can be applied to fields such as tissue regeneration, pharmaceuticals (topical and sustained drug delivery), antimicrobial, orthopedics, regenerative medicine, cosmetic surgery, supplements, and cosmetics. The injectable gel can be used in forms such as subcutaneous injection drugs, spray formulations, coating materials, cell regeneration scaffolds, drug delivery matrices, cosmetic surgery matrices, and surgical adhesion prevention materials. The injectable gel may also be a redox injectable gel (RIG).

[0013] <1-1. Triblock Copolymer> The first triblock copolymer has a structure represented by polycation-block-polyethylene glycol-block-polycation. The second triblock copolymer has a structure represented by polyanion-block-polyethylene glycol-block-polyanion. In this specification, the connection symbol "-block-" means that the segments connected by this "-block-" each constitute a block, and will be abbreviated as "-b-" below. That is, in the first triblock copolymer, it can be said that a polycation segment, a PEG segment, and a polycation segment are connected in this order. The second triblock copolymer has a structure represented by a polyanion segment, a PEG segment, and a polyanion segment.

[0014] The PEG segment is -(OCH2CH2) m It can be represented as -, where m is preferably an integer between 20 and 800, more preferably between 30 and 500, and even more preferably between 40 and 400.

[0015] Examples of polymers constituting the polycation segment include polymers having substituents in their side chains that can be protonated in water. The main skeleton of the polymer is not limited, but examples include polyamino acids, polymethacrylic acid derivatives, polyacrylic acid derivatives, and polystyrene derivatives. Examples of substituents include primary amino groups and secondary amino groups, specifically amino groups and imidazole groups. Examples of polyamino acids include polylysine, polyarginine, polyornithine, polyhistidine, and polytryptophan.

[0016] The polycation segment may have a cyclic nitroxide radical as part of the pendant group. Examples of cyclic nitroxide radicals include 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidinedine-3-oxyl-2-yl, and 2,4,4-trimethylimidazolindinine-3-oxyl-2-yl. The cyclic nitroxide radical is o- or p-phenylene-C 1~6 Alkylene-NH-(C) 1~6 The alkylene may be linked to the polycation segment via q-(where q is 0 or 1).

[0017] Polymers that constitute polyanion segments include polyacrylic acid, polymethacrylic acid, polysulfonic acid, polystyrene sulfonic acid, poly(benzoate) vinyl, polyanionic polysaccharides, and anionic proteins. Examples of polyanionic polysaccharides include chondroitin sulfate, carrageenan, heparin, carboxymethyl dextran, xanthan gum, and hyaluronic acid. Examples of anionic proteins include polyaspartic acid and polyglutamic acid.

[0018] The degree of polymerization (the number of repeating monomer units) of the polycation segment or the polyanion segment is not limited from the viewpoint of the stability of the PIC micelles in an aqueous medium, but is preferably an integer of 10 to 200, more preferably 15 to 150, still more preferably 15 to 100, and particularly preferably 20 to 100.

[0019] Each segment can be linked by a linking group. The linking group can be a divalent organic group that is not limited as long as it does not adversely affect the formation of PIC. Examples of the linking group include -O-(CH2) a -NH-, -O-(CH2) a -O-, -(CH2) a -NH-, -(CH2) a -O-,

[0020]

Chemical formula

[0021] etc. Here, a is an integer of 1 to 6, preferably 1 to 3.

[0022] The terminal of the triblock copolymer, that is, the terminal opposite to the terminal linked to the PEG segment in the polycation segment or the polyanion segment, may be, for example, H, an amino group, a linear or branched C 1~6 alkyl group, a phenylthiocarbonylthio group, a C 1~6 alkyloxyalkylthiocarbonylthio group, a C 1~6 alkyloxythiocarbonylthio group or a sulfanyl group, etc. The C 1~6 alkyl group, a phenylthiocarbonylthio group, a C 1~6 alkyloxyalkylthiocarbonylthio group, a C 1~6 alkyloxythiocarbonylthio group may be unsubstituted or substituted, and when substituted, the substituent is a C 1~4 alkyl group, a C 1~4 alkyloxy group, a hydroxyl group, a carboxyl group, a cyano group, a nitro group, a halogen atom group, or a mono- or di-C1~4 It may be an alkylamino group.

[0023] The molecular weight distribution of the triblock copolymer does not need to be limited during gelation, but may be, for example, 1.01 to 1.25, 1.01 to 1.20, 1.01 to 1.15, or 1.01 to 1.10.

[0024] The method for producing triblock copolymers is not particularly limited, and methods described in Patent Document 1, International Publication No. 2015 / 118993, International Publication No. 2014 / 199982, Ishii et al., Macromolecules 2015, 48, 3088-3094, Long Binh Vong et al., Biomaterials 167 (2018) 143-152, Saita et al., Biomaterials 76 (2016), 292-301, Nakagawa et al., Biomaterials 69 (2015) 165-173, Min Ley Pua et al., J. Control. Release 172 (2013), 914-920, etc., can be referenced.

[0025] <1-2. Polyanions and Polycations> As the polyanion to be combined with the first triblock copolymer, the polymers exemplified as constituting the polyanion segment of the second triblock copolymer can be used. Furthermore, as the polycation to be combined with the second triblock copolymer, the polymers exemplified as constituting the polycation segment of the first triblock copolymer can be used.

[0026] The molecular weights of these polyanions and polycations are not limited and the optimal values ​​vary depending on the type of polymer. For example, in the case of polyacrylic acid, the Mn (number-average molecular weight) is preferably 200 to 1,000,000, more preferably 500 to 1,000,000, and even more preferably 1,000 to 10,000. In the case of polyanionic polysaccharides, such as chondroitin sulfate, the Mn or Mw (weight-average molecular weight) is preferably 500 to 1,000,000, more preferably 1,000 to 100,000. In the case of anionic polypeptides, such as polyaspartic acid, the Mn or Mw is preferably 500 to 1,000,000, more preferably 1,000 to 100,000.

[0027] <1-3. Polyion Complex> PICs are formed in an aqueous medium via electrostatic interactions between the polycation segment of a first triblock copolymer and the polyanion (as described in (a) above). These PICs may exist as nanosized polymer micelles having a core formed via electrostatic interactions between the polycation segment and the polyanion, and a shell formed from the PEG segment of the first triblock copolymer. Since these polymer micelles can be observed in particulate form, they are also referred to herein as self-assembled nanoparticles. Similar PICs are also formed by electrostatic interactions between the polyanion segment of a second triblock copolymer and the polycation (as described in (b) above). Furthermore, similar PICs are also formed by electrostatic interactions between the polycation segment of a first triblock copolymer and the polyanion segment of a second triblock copolymer (as described in (c) above).

[0028] The specific combinations of the first triblock copolymer with a polyanion, the second triblock copolymer with a polycation, and the first triblock copolymer with the second triblock copolymer are not particularly limited. In the examples, measurement results are shown for combinations of a triblock copolymer having a polylysine segment with polystyrene sulfonic acid and a triblock copolymer having a cyclic nitroxide radical with polyacrylic acid, but combinations of a triblock copolymer having a polylysine segment with polyacrylic acid and a triblock copolymer having a cyclic nitroxide radical with polystyrene sulfonic acid are also possible.

[0029] Examples of the aqueous medium include pure water, deionized water, buffered solutions thereof, and solutions containing water-soluble organic solvents. Examples of water-soluble organic solvents include N,N-dimethylformamide, dimethyl sulfoxide, alcohols such as methanol and ethanol, acetone, and tetrahydrofuran.

[0030] For manufacturing methods of PICs, refer to methods described in, for example, Patent Document 1, International Publication No. 2015 / 118993, International Publication No. 2014 / 199982, Ishii et al., Macromolecules 2015, 48, 3088-3094, Long Binh Vong et al., Biomaterials 167 (2018) 143-152, Saita et al., Biomaterials 76 (2016), 292-301, Nakagawa et al., Biomaterials 69 (2015) 165-173, Min Ley Pua et al., J. Control. Release 172 (2013), 914-920, etc.

[0031] <1-4. Inorganic Nanoparticles> The materials that make up inorganic nanoparticles are not particularly limited, and examples include silica (silica gel), titanium dioxide, gold, and silver.

[0032] The average particle size of the inorganic nanoparticles is on the order of nanometers, preferably 100 nm or less, more preferably 70 nm or less, even more preferably 50 nm or less, even more preferably 20 nm or less, particularly preferably 10 nm or less, and most preferably 6 nm or less. Furthermore, the average particle size of the inorganic nanoparticles is preferably 1 nm or more, and more preferably 4 nm or more. The average particle size can be determined from the volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering measurement. Alternatively, the average particle size may be measured by transmission electron microscopy.

[0033] The inorganic nanoparticles are compounded with the self-assembled nanoparticles and are presumed to be incorporated into the interior of the self-assembled nanoparticles, for example. For example, particles compounded with silica nanoparticles and self-assembled nanoparticles are also referred to as silica-compounded self-assembled nanoparticles in this specification. The average particle size of the self-assembled nanoparticles compounded with inorganic nanoparticles may be 3000 nm or less, 2500 nm or less, 2000 nm or less, 1500 nm or less, or 1000 nm or less. Furthermore, the average particle size of the self-assembled nanoparticles compounded with inorganic nanoparticles may be 100 nm or more, 200 nm or more, or 300 nm or more. The average particle size can be determined from the volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering measurement.

[0034] A method for producing a gel manufacturing composition according to one embodiment of the present invention includes a step of mixing the polyion complex described in (a), (b), or (c) above with inorganic nanoparticles. For example, a gel manufacturing composition can be obtained by mixing a solution or dispersion containing PIC with a solution or dispersion containing inorganic nanoparticles. Examples of solvents contained in these solutions or dispersions include the aqueous media described above. When mixing, a mixer may be used for stirring.

[0035] [2. Gel] A gel according to one embodiment of the present invention is obtained by gelling the above-described gel manufacturing composition. A method for manufacturing a gel according to one embodiment of the present invention includes the steps of obtaining a gel manufacturing composition by the above-described method for manufacturing a gel manufacturing composition, and gelling the gel manufacturing composition. Examples of gelling methods include increasing the temperature and / or ionic strength. For example, examples include heating the gel manufacturing composition to 35-45°C and / or contacting the gel manufacturing composition with physiological saline solution with an NaCl concentration of 100-200 mM.

[0036] [3. Gel manufacturing kit] A gel manufacturing kit according to one embodiment of the present invention comprises a first agent containing the polyion complex described in (a), (b), or (c) above, and a second agent containing inorganic nanoparticles. By mixing the first agent and the second agent using this gel manufacturing kit, the polyion complex and inorganic nanoparticles are mixed and gelled, thereby producing a gel.

[0037] The PIC may be in the form of a solution or dispersion in the first component, or it may be in a dry state. The inorganic nanoparticles may be in the form of a solution or dispersion in the second component, or it may be in a dry state. When mixing, it is preferable that the first and second components are mixed in the form of a solution or dispersion containing the aqueous medium described above.

[0038] The first and second components may be contained in separate containers. In this specification, the container containing the first component will be referred to as the first container, and the container containing the second component as the second container. The first and second containers may be independent and separate containers, or they may be integrated into a single container. When the first and second containers are integrated into a single container, it means that they are configured as a single container comprising a first space containing the PIC and a second space containing inorganic nanoparticles. The gel manufacturing kit may also include another container (space) containing the aqueous medium described above for manufacturing the solution or dispersion.

[0039] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0040] One embodiment of the present invention may include the following configuration: <1> A gel-making composition comprising a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. <2> <1> A gel obtained by gelling a gel manufacturing composition. <3> A method for producing a gel composition, comprising the step of mixing a polyion complex with inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. <4> <3> A method for producing a gel, comprising the steps of: obtaining a gel-producing composition by the method for producing a gel-producing composition described in [reference]; and gelling the gel-producing composition. <5> A gel manufacturing kit comprising a first agent containing a polyion complex and a second agent containing inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. [Examples]

[0041] An embodiment of the present invention is described below. In the following, [polymer] refers to the polymer concentration in the solution, [NaCl] refers to the sodium chloride concentration in the solution, [PB] refers to the phosphate buffer concentration in the solution, and [ST-XS] refers to the ST-XS (silica nanoparticle) concentration in the solution.

[0042] [Material property evaluation] The number-average molecular weight (Mn), weight-average molecular weight (Mw), and molecular weight distribution of each polymer were measured. Two polystyrene gel columns (Tosoh Corporation, TSKgel GMHHR-M; exclusion limit: molecular weight (MW) = 4.0 × 10⁻¹⁰) were connected to a pump (JASCO, PU-4180). 6 Gel permeation chromatography (GPC) was performed in N,N-dimethylformamide (DMF) at 40°C (flow rate: 0.40 mL / min) using a differential refractive index (RI) detector (JASCO, RI-2031) and a UV / Vis detector (JASCO, UV-4075) in a column with particle size: 5 μm; pore size: data not available; inner diameter 7.8 cm × 30 cm). The column was calibrated using 18 standard poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG) samples (Merck; Mp=238-1,180,000). 1 The H nuclear magnetic resonance (NMR) spectrum was obtained using an AVANCE-600 NMR spectrometer (Bruker) at 600 MHz. 1 The system was operated at H) and acquired in CDCl3 or DMSO-d6 at room temperature (22-23°C). Dynamic light scattering (DLS) measurements were performed at 37°C using a Zetasizer Nano ZSP (Malvern) equipped with a He-Ne laser (λ=633nm). The measurement angle was 173°, and the data were analyzed using the non-negative least squares method (NNLS).

[0043] [Example 1: Synthesis of Z-protected L-lysine-N-acid anhydride (NCA-Lys(Z))] L-lysine (HOCOCH((CH2)4NHCOOCH2C6H5)NH2;H-Lys(Z)-OH) (25.2g, 89.9 mmol; 1.0 eq.), which has a benzyloxycarbonyl group in its side chain, and triphosgene (18.2g, 61.3 mmol; approximately 0.67 eq.) were dissolved in α-pinene (36 mL, 227 mmol; 2.5 eq.) and tetrahydrofuran (THF; 252 mL), and the mixture was stirred at 50°C for 3 hours. After the resulting reaction solution was cooled to room temperature, it was gradually added to hexane (600 mL) to obtain a white precipitate. The white precipitate was washed with hexane and recovered by vacuum filtration. The recovered white compound was redissolved in THF / acetone / isopropanol (IPA) (approximately 10 / 10 / 1 (v / v / v)), and reprecipitation in hexane was repeated two more times. Finally, the white precipitate recovered by vacuum filtration was dried under reduced pressure to obtain the target product NCA-Lys(Z) (yield 16.4g, yield 59%).

[0044] [ka]

[0045] [Example 2 H-(HNCH((CH2)4NHCOOCH2C6H5)CO) n NH-CH2CH2(OCH2CH2) m -NH(COCH((CH2)4NHCOOCH2C6H5)NH) n -H synthesis) PEG macroinitiator H2N-(OCH2CH2) n -NH2 (Mn=10,000, 25.1g, 2.51 mmol) was dissolved in N,N-dimethylformamide (DMF; 44.8 mL) and tetralin (0.3 mL). Furthermore, a 1000 mM monomer solution was prepared by dissolving NCA-Lys(Z) synthesized in Example 1 in DMF. The monomer solution (70.4 mL, 70.4 mmol) was mixed with H2N-(OCH2CH2) nThe mixture was added to a DMF solution of -NH2 and mixed, and reacted at 45°C for 4 days. The resulting reaction solution was directly added to hexane / IPA (=1 / 1(v / v)) to precipitate, and the precipitate was recovered by centrifugation. The recovered precipitate was redissolved in DMF, and the reprecipitation and purification by centrifugation were repeated two more times. The target product (PLys(Z)-b-PEG-b-PLys(Z)) was recovered by vacuum drying (30.5g, n=7.7(P2), Figure 1b). Polymers n=2.7(P1), 19(P3), and 54(P4) were synthesized similarly (Figures 1 and 2). The obtained polymers are abbreviated as P1, P2, P3, and P4.

[0046] [ka]

[0047] [Example 3 H-(HNCH((CH2)4NH3)CO) n NH-CH2CH2(OCH2CH2) m -NH(COCH((CH2)4NH3)NH) n -H synthesis) P2 (30.5 g, 33.6 mmol (Lys(Z)); 1.0 eq.) synthesized in Example 2 was dissolved in trifluoroacetic acid (TFA) (300 mL) and cooled in an ice bath. Hydrogen bromide (HBr) acetic acid solution (28%; 61 mL, 337 mmol; approximately 10 eq.) was slowly added dropwise. The mixture was stirred overnight to allow the reaction to proceed. The resulting reaction solution was directly transferred to a dialysis membrane (MWCO (molecular weight cutoff) = 3500 Da), dialyzed in methanol / Na2CO3 aqueous solution (= 1 / 1 (v / v)) for 3 days and in water for 4 days, and then the target product (PLys-b-PEG-b-PLys) was recovered by freeze-drying (19.6 g, deprotection rate 100%). P1, P3, and P4 were deprotected in the same manner (Figure 2b).

[0048] [ka]

[0049] [Example 4 Preparation of self-assembled nanoparticles 1: Formation of a polyion complex with poly(styrene sulfonic acid)] PLys-b-PEG-b-PLys synthesized in Example 3 were dissolved in dimethyl sulfoxide (DMSO) / TFA = 20 / 1 (v / v) ([polymer] = 5 mg / mL). Sodium poly(styrene sulfonate) (NaPSS; Sigma-Aldrich, Mw: approximately 75000) was dissolved in phosphate buffer (pH = 7.4) ([polymer] = 5 mg / mL). The NaPSS phosphate buffer was slowly added dropwise to the PLys-b-PEG-b-PLys solution while stirring to prepare a mixed solution. This mixed solution was directly transferred to a dialysis membrane (MWCO = 3500 Da), dialyzed against water for 4 days, and then concentrated under reduced pressure to obtain self-assembled nanoparticles (Nano Lys / PSS A concentrated solution of [polymer] was obtained. This concentrated solution was diluted with water, and dynamic light scattering (DLS) measurements were performed to confirm the formation of self-assembled nanoparticles of 10-43 nm (Figures 3, 4; [polymer] = 10 mg / mL).

[0050] [Example 5 Preparation of Self-Assembled Nanoparticles 2: Composite Formation of Silica Nanoparticles] After diluting the concentrated solution of self-assembled nanoparticles prepared in Example 4 with water, an aqueous solution of dispersed silica nanoparticles was added and immediately stirred for 5 minutes using a vortex mixer to form silica-compounded self-assembled nanoparticles (Nano Lys / PSS / SiO A solution was prepared ([polymer]=40 mg / mL). Snowtex® (Nissan Chemical Corporation; Snowtex® XS (ST-XS), Snowtex® 30 (ST-30)) was used as the silica nanoparticle. The size of ST-XS was 4-6 nm, and the size of ST-30 was 10-15 nm. For DLS measurement, the solution was diluted 40 times with water before measurement (Figures 7, 8; [polymer]=1.0 mg / mL).

[0051] [Example 6: Rheological Measurement] The gelation behavior was evaluated using an Anton Paar rheometer (MCR302). In this experiment, the storage modulus (G'), loss modulus (G''), and complex viscosity ([η) were evaluated. * The storage modulus and loss modulus were measured to evaluate the gelation process and gel properties. The storage modulus and loss modulus indicate solid and liquid properties, respectively. For example, when the storage modulus is higher than the loss modulus (G'>G''), the material exhibits mainly solid properties. Furthermore, the gelation point can be determined at the intersection where both moduli are the same value (G'=G''). The self-assembled nanoparticle aqueous solutions ([polymer]=40 mg / mL; 100 μL) obtained in Examples 4 and 5 were placed on a stage with a distance of 0.2 mm to the plate. A 20 mm parallel plate was used. The measurement frequency was fixed at 1 Hz, and the temperature was varied from 15°C to 45°C, followed by continuous cooling from 45°C to 15°C. The temperature change rate was 1°C / min.

[0052] [Example 7: Influence of ionic strength on temperature dependence of elastic modulus and viscosity measurements] Sodium chloride (NaCl) is converted into PLys-b-PEG-b-PLys(P4) / PSS self-assembled nanoparticles (Nano Lys / PSS (P4)) The polymer was dissolved in an aqueous solution ([polymer]=40 mg / mL, [NaCl]=0, 150, 500 mM). According to Example 6, the aqueous solution was measured, and gelation occurred at 25.9°C and 24.7°C under NaCl concentrations of 150 mM and 500 mM, respectively. The resulting gel did not return to its initial modulus even after cooling, demonstrating irreversible gelation behavior. The modulus (G') after gelation decreased slightly as the NaCl concentration increased, but remained around several hundred Pa under all conditions (Figure 5). Furthermore, these results suggest that Nano Lys / PSS (P4) was shown to gel depending on temperature and ionic strength.

[0053] [Example 8: Influence of the degree of polymerization of lysine (DP(Lys)) on the temperature dependence of elastic modulus and viscosity measurements] The NaCl concentration was fixed at 150 mM (physiological saline concentration), and PLys-b-PEG-b-PLys / PSS self-assembled nanoparticles (Nano Lys / PSS The aqueous solutions were measured according to Example 6, and the effects of P3 and P4 were investigated ([polymer]=40 mg / mL, [NaCl]=150 mM). Gelation occurred in both P3 and P4 (Figure 6).

[0054] [Example 10: Influence of silica nanoparticles on the temperature dependence of elastic modulus and viscosity measurements] The silica-composite nanoparticles prepared in Example 5 (Nano Lys / PSS / SiO The elastic modulus was measured according to Example 6. In this case, Snowtex® series silica nanoparticles were used. As a result, when silica nanoparticles were compounded, the elastic modulus after gelation improved compared to when silica nanoparticles were not compounded (Figures 9, 10). In particular, when Snowtex® XS (ST-XS) was used, a high elastic modulus exceeding 10 kPa was achieved after gelation (Figures 9, 10).

[0055] [Example 11: Synthesis of Cl-PEG-Cl] A 500 mL round-bottom flask equipped with a three-way stopcock contains HO-(CH2CH2) n -OH (Mn=10,000, 50.0g, 5.00 mmol) was added and the mixture was dried overnight under reduced pressure at 110°C. After lowering the liquid temperature to 65°C, THF (200 mL) was added to dissolve the contents of the flask. Butyllithium solution (20.0 mmol, 12.5 mL, 1.6 M hexane solution) was gradually added under a nitrogen atmosphere to activate the hydroxyl groups. Subsequently, dichloro-p-xylene (17.5 g, 0.1 mol) was added and the mixture was reacted at 60°C for 4 days. The resulting reaction mixture was precipitated in IPA cooled to 4°C, and the precipitate was recovered by centrifugation. The precipitate was again dissolved in 20 mL of methanol, and reprecipitation was performed in cooled IPA. The reprecipitation and centrifugation steps were repeated four more times. The target product (Cl-PEG-Cl) was recovered by drying under reduced pressure.

[0056] [Example 12: Synthesis of Grignard Reagent] THF (10 mL) was added to a 50 mL round-bottom flask equipped with a three-way stopcock, and the mixture was cooled in an ice bath at 0°C. Under a nitrogen atmosphere, carbon disulfide (3.75 mL, 62.0 mmol) and phenylmagnesium bromide (16.0 mmol, 5.19 mL, 3.0 M diethyl ether solution) were added, and the mixture was reacted overnight in the ice bath. The synthesized Grignard reagent was used without purification.

[0057] [Example 13: Synthesis of macro-RAFT agent (CTA-PEG-CTA)] In a 300 mL round-bottom flask equipped with a three-way stopcock, the Cl-PEG-Cl (Mn (size exclusion chromatography (SEC)) = 9,300, 40.0 g, 3.9 mmol) synthesized in Example 12 was added and dried overnight under reduced pressure at 110°C. After lowering the liquid temperature to 65°C, 140 mL of THF was added to dissolve the contents of the flask. Under a nitrogen atmosphere, the Grignard reagent synthesized in Example 12 was gradually added and reacted at 40°C for 24 hours. The resulting reaction solution was precipitated in IPA cooled to 4°C, and the precipitate was recovered by centrifugation. The precipitate was again dissolved in 20 mL of methanol, and reprecipitation was performed in cooled IPA. The reprecipitation and centrifugation steps were repeated three more times. The target product (CTA-PEG-CTA) was recovered by reduced-pressure drying.

[0058] [Example 14: Synthesis of PCMS-b-PEG-b-PCMS] In a 500 mL round-bottom flask equipped with a three-way stopcock, CTA-PEG-CTA (Mn(SEC)=10,100,35.0 g, 3.30 mmol) synthesized in Example 13 and azobisisobutyronitrile (AIBN) (547 mg, 3.30 mmol) were added, and the mixture was degassed by reducing the pressure for 30 minutes. Toluene (350 mL) was added under a nitrogen atmosphere to dissolve the contents of the flask. p-chloromethylstyrene (32.8 mL, 0.230 mol), purified by vacuum distillation, was added, and the mixture was reacted at 60°C for 24 hours. The resulting reaction mixture was precipitated in methyl t-butyl ether (2 L), and the precipitate was collected by vacuum filtration. The collected precipitate was dissolved in acetone (20 mL) and reprecipitated in methyl t-butyl ether (2 L). The precipitate was collected by vacuum filtration and dried under reduced pressure overnight. In a 500 mL round-bottom flask equipped with a reflux tubing, the dried precipitate, AIBN (14.5 g, 89.8 mmol), and ethyl acetate (300 mL) were added and dissolved. After degassing by nitrogen bubbling for 30 minutes, the reaction was carried out at 80°C for 12 hours. The reaction mixture was precipitated in methyl t-butyl ether (2 L), and the precipitate was recovered by vacuum filtration. The recovered precipitate was dissolved in acetone (20 mL) and reprecipitated in methyl t-butyl ether (2 L). The target product (PCMS-b-PEG-b-PCMS) was recovered by vacuum drying.

[0059] [Example 15: Synthesis of PMNT-b-PEG-b-PMNT] PCMS-b-PEG-b-PCMS (Mn(SEC)=13,000, 8.27g, 62.7mmol) synthesized in Example 14 was added to a 200mL round-bottom flask equipped with a three-way stopcock, and dried under reduced pressure for 30 minutes. After adding DMF (80mL) to dissolve the contents of the flask, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-aminoTEMPO) (10.0g, 58.4mmol) dissolved in 20mL of DMF was added, and the mixture was reacted at 50°C for 24 hours. The resulting reaction solution was precipitated in IPA cooled to 4°C, and the precipitate was recovered by centrifugation. The precipitate was again dissolved in 10mL of acetone, and reprecipitation was performed in cooled IPA. The reprecipitation and centrifugation steps were repeated four more times. The target product (PMNT-b-PEG-b-PMNT) was recovered by drying under reduced pressure.

[0060] [Example 16 Preparation of self-assembled nanoparticles 3: Formation of a polyion complex of PMNT-b-PEG-b-PMNT and polyacrylic acid] PMNT-b-PEG-b-PMNT and polyacrylic acid (PAAc; Fujifilm Wako, Mn: approximately 5,000) synthesized in Example 15 were each dissolved in phosphate buffer (pH=6.2, 100 mM) ([polymer]=10 mg / mL). The PMNT-b-PEG-b-PMNT solution was gradually added dropwise to the PAAc phosphate buffer while being slowly stirred to prepare a mixed solution. Dynamic light scattering measurements were performed to confirm the formation of 62 nm self-assembled nanoparticles (Figure 11 (RIG)).

[0061] [Example 17: Rheological evaluation of redox-injectable gel (RIG)] The mixed solution prepared in Example 16 was concentrated using a centrifugal evaporator to prepare a RIG ([polymer]=60 mg / mL, [PB]=600 mM). After distillation and dilution with deionized water ([polymer]=40 mg / mL, [PB]=400 mM), rheological measurements were performed according to Example 6, and gelation occurred at 25.1°C. The resulting gel did not return to its initial modulus even after cooling, confirming irreversible gelation behavior (Figure 12).

[0062] [Example 18: Rheological evaluation of silica nanoparticle composite RIG (RIG / Snowtex(registered trademark) ST-XS)] Snowtex® XS (ST-XS) was added to the RIG ([polymer]=60 mg / mL, [PB]=600 mM) prepared in Example 17, and the mixture was thoroughly stirred in a vortex mixer to prepare silica-compounded self-assembled nanoparticles RIG / ST-XS ([polymer]=40 mg / mL, [PB]=400 mM, [ST-XS]=5.6 wt%). Rheological measurements were performed according to Example 6, and a high modulus of elasticity exceeding 10 kPa was achieved after gelation (Figure 11 (RIG / ST-XS), Figure 12). [Industrial applicability]

[0063] One aspect of the present invention can be used, for example, in the production of injectable gels.

Claims

1. It contains a polyion complex and inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) A polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. A composition for manufacturing gels.

2. A gel obtained by gelling the gel manufacturing composition described in claim 1.

3. The process includes a step of mixing a polyion complex with inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) A polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. A method for producing a gel composition.

4. A step of obtaining a gel manufacturing composition by the method for manufacturing a gel manufacturing composition described in claim 3, A method for producing a gel, comprising the step of gelling the aforementioned gel-producing composition.

5. The solution comprises a first agent containing a polyion complex and a second agent containing inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) A polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. This is a gel manufacturing kit.

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